An essential requirement for effective genetic engineering of bacteria and other cells propagated in cell cultures is the capacity to select the cells with a specific genotypic alteration. The most common selection strategy in recombinant DNA technology is to include a selection marker in the cloning vector or plasmid. A selection marker can be a cloned gene or a DNA sequence, which allows the separation of the host cells containing the selection marker from those not containing it. The selection marker together with a suitable selection medium maintains the cloning vector in the cells. Otherwise, since the replication of plasmids is an energetic burden for the bacterial host, in a growing culture the bacteria, which have lost the plasmid, would have a growth advantage over the cells with the plasmid.
For most purposes, an antibiotic resistance gene is a commonly used selection marker. However, for the production of recombinant therapeutics, where the goal is to generate a product, such as a DNA vaccine, in high yield for administration in patients, the use of antibiotic resistance genes presents problems: the spread of antibiotic resistant pathogens is a serious worldwide problem [Levy, S. B., J. Antimicrob. Chemother. 49 (2002) 25-30]. Therefore the antibiotic resistance genes cannot have extensive use in the pharmaceutical industry, and for instance, according to the regulations of the U.S. Food and Drug Administration, no antibiotic resistance genes are allowed in experimental DNA vaccines entering the third phase.
Alternatively, antibiotic-free selection systems have been suggested. Such antibiotic-free selection systems include bacterial toxin-antitoxin systems [Engelberg-Kulka, H. and Glaser, G., Annu Rev Microbiol 53 (1999) 43-70], genes responsible for resistance against heavy metals, such as tellurium [Silver, S, and Phung, L. T., Annu Rev Microbiol 50 (1996) 753-789], and systems, in which the plasmid encodes a gene complementing a host auxotrophy [Wang, M. D., et al., J. Bacteriol. 169 (1987) 5610-5614].
US Patent Application 2000/0014476 A1 generally discloses, inter alia, the use of a non-antibiotic selection marker, which may be a gene whose product is necessary for the metabolism of the cell under certain culturing conditions, such as a catabolism gene, which makes it possible for the cell to assimilate a certain substance present in the culture medium (specific carbon or nitrogen source) etc. No specific examples of such suitable genes are given. This approach is not necessarily applicable for commercial production, since the deletion an essential component, such as an amino acid or a carbon source, from the growth medium reduces the yield, which is not desirable. Additionally, the manipulation of the growth medium in terms of omitting an essential nutrient may considerably increase the cost of the growth medium, since commercially available nutrient mixtures must be replaced by individual nutrients.
For commercial therapeutic purposes it would be of advantage to use a gene, which is not essential for the growth of the host but whose manipulation still affects the growth in selected circumstances. Additionally, in view of the therapeutic use, it would be of advantage to use a gene, whose deletion leads to accumulation of compounds, which are toxic to the host cell but not toxic to mammalians, including humans. Also it would be of advantage to use smaller genes, which in turn would allow the construction of smaller plasmids for which the energy consumption for replication is smaller and thus the growth rate of bacterial culture and plasmid yield are improved.